BSC 450: Chapter 4

The Three-Dimensional Structure of Proteins

4.1, Overview of Protein Structure

Conformation: the spatial arrangement of atoms in a protein or part of a protein

 Any structural state it can achieve without breaking covalent bonds
 Change can occur by rotation about a single bond
 A few generally predominate

Native State: proteins in any of their functional, folded conformations

A Protein’s Conformation Is Stabilized Largely by Weak Interactions

 Stability: the tendency to maintain a native conformation
o Native proteins are only marginally stable
o A polypeptide can assume countless configurations, so the unfolded
state is characterized by a high degree of conformational entropy
 Entropy and hydrogen bonding with water  tends to remain unfolded
o Chemical interactions can counteract this effect
 Disulfide (covalent) bonds
 Most proteins do not have disulfide bonds due to the
highly reducing environment inside the cell
 In eukaryotes, disulfide bonds are primarily found in
extracellular proteins
 Uncommon in bacterial proteins, except those adapted to
high temperatures (archaea)
 Weak (noncovalent) interactions
 hydrogen bonds, hydrophobic effect, ionic interactions
 The associations of multiple polypeptides to form their quaternary structures
relies on weak interactions
 Covalent bonds such as disulfide bonds are much stronger than individual
weak interactions, but because they are so numerous, the weak interactions
predominate as a stabilizing force in the proteins
o Protein conformation with the lowest free energy is the one with the
maximum number of weak interactions
 The stability of a protein is not simply the sum of the free energies of
formation of the many weak interactions within it
 o For every hydrogen bond formed, a hydrogen bonds between the
group and water is broken
o The net stability contributed, or the difference in free energies may be
close to zero
o Ionic interactions may be either stabilizing or destabilizing
 The hydrophobic effect predominates in the contribution of weak
interactions to protein stability
o When water surrounds a hydrophobic molecule, it creates a solvation
layer around the molecule, which correlates with an unfavorable
decrease in entropy
 But when nonpolar groups cluster, the extent of the solvation
layer decreases, resulting in an increase in entropy
o Hydrophobic amino acid side chains tend to cluster in the protein’s
interior, forming a hydrophobic protein core
 The formation of hydrogen bonds in a protein is driven by the same entropic
effect
o A solvation layer forms to some extent even around polar molecules
o The release of structured water as intramolecular associated form
provides an entropic driving force for folding
o Most of the net change in free energy as nonpolar amino acid side
chains aggregate is derived from the increased entropy in the
surrounding aqueous solution from the burial of hydrophobic surfaces
 This counterbalances the large loss of conformational entropy
as a polypeptide is constrained into its folded conformation
 The hydrophobic effect is clearly important in stabilizing conformation,
interior of structured protein is generally a densely packed core of
hydrophobic amino acids
o Hydrogen-bonding groups without partners in the hydrophobic core
can be destabilizing
 Interaction of oppositely charged groups that form an ions pair or salt bridge,
can have a stabilizing or destabilizing effect on protein structure
o Charged amino acid side chains interact with water and salts when
unfolded
 Salt bridges can provide significant stabilization to a protein
o Ionic interactions limit flexibility, provide uniqueness to structure
 Van der Waals interactions (dipole-dipole interactions involving the
permanent electric diploes, transient dipoles derived from fluctuation in
electron clouds, and dipoles induced by interaction of one atom that has a
 permanent or transient dipole) provide an attractive intramolecular force that
operates over a limited intermolecular distance
o In a well packed protein, the effect of many of these interactions can
be substantial
 Two simple rules
o 1. Hydrophobic resides are largely buried in the protein interior, away
from water
o 2. The number of hydrogen bonds and ionic interactions is
maximized, thus reducing the number of hydrogen-bonding and ionic
groups that are not paired with a suitable partner
o Proteins within membranes and intrinsically disordered proteins
follow different rules
 Reflecting their particular function or environment
 Weak interactions are still critical structural elements

The Peptide Bond is Rigid and Planar

 The alpha carbons of adjacent amino acid residues are separated by three
covalent bonds as Ca-C-N-Ca
o The C-N bond is shorter than normal
o Atoms associated are coplanar
o Indicates a resonance, partial sharing of two electron pairs between
the carbonyl oxygen and the amide nitrogen
 Oxygen has partial negative charge, nitrogen has partial
positive, setting up a small electric dipole
o The six atoms of the peptide group lie in a single plane
 The oxygen atom trans to the hydrogen of the amide nitrogen
o The C-N bonds do not rotate freely due to their partial double bond
character
o The backbone of a polypeptide can be seen as a series of rigid planes
with consecutive planes sharing a common point of rotation at Ca
 The rigid peptide bonds limit the range of conformations
possible for a polypeptide chain
 Peptide conformation is defined by three dihedral angles, phi, psi, and
omega, reflecting rotation about each of the three repeating bonds in the
peptide backbone
o Dihedral angle: the angle at the intersection of two planes
 Defined by bond vectors int eh peptide backbones
 Two successive bond vectors describe a plane
 Three successive bond vectors describe two planes
  The angle between these planes is what we measure to describe
peptide conformation
o Phi and psi can have any value between -180 and +180 degrees
 Many values prohibited by steric interference between atoms in
backbone and amino acids side chains
 Cannot both be 0, merely a reference point
o Ramachandran plot: psi plotted versus phi, allowed values become
evident
 Often used to test the quality of three-dimensional protein
structures that are deposited

4.2, Protein Secondary Structure

Secondary structure: any chosen segment of a polypeptide chain

 Describes the local spatial arrangement of its main-chain atoms without
regard to the positioning of its side chains or relationship to other segments

The Alpha Helix is a Common Protein Secondary Structure

 The alpha helix: a helical structure discovered by Pauling and Corey
o Polypeptide backbone is tightly wound around imaginary axis through
the middle of the helix
o The R groups of the amino acid residues protrude outward from the
helical backbone
o The repeating unit is a single turn of the helix, extends about 5.4 A
along axis
o Each helical turn includes 3.6 amino acid residues
 5.4 A = 3.6 amino acid residues
 1.5 A per reside
 The a-helical segments in proteins often deviate slightly from dihedral
angles, and can bend and kink in the axis
o Left and right handed variants
 The right-handed a helix is the common form
 Extended left hand a helices are less stable and have not been
observed in proteins
 About one forth of all amino acid resides in proteins are found in a helices,
the exact fraction varying from one protein to another
 The structure is stabilized by a hydrogen bond between the hydrogen atom
attached to the electronegative nitrogen atom and the electronegative
 carbonyl oxygen of the fourth amino acid on the a-terminal side of that
peptide bond
o Every peptide bonds participates in hydrogen bonding
o Each successive turn is held to adjacent turns by three to four
hydrogen bonds (conferring stability)
o The ends three or four carbonyl or amino groups cannot participate in
the patter, so they are exposed to the solvent to form hydrogen bonds

Amino Acid Sequence Affects Stability of the a Helix

 Not all polypeptides can form the a -helix
o Each amino acid reside has an intrinsic propensity to form an a helix
 Reflects R group properties and how they affect main chain
atoms to take up phi and psi angles
 Alanine shows the greatest tendency to form a helices
o Position of amino acids residue relative to its neighbors is also
important
 Long block of Glu resides will not form helix
 Negatively charged carboxyl groups repel each other
 Adjacent Lys or Arg residues with positive R groups will also
repel each other
 The bulk shape of Asn, Ser, Thr, and Cys resides can also
destabilize an a helix if they are close together
o Pro or Gly resides have the least proclivity to form a helices
 In Pro residues, the nitrogen is part of a rigid ring and rotation
about the N-Ca bond is not possible, introducing a destabilizing
kink in the helix
 Pro resides also have no hydrogen in nitrogen atom to produce
hydrogen bonds
 Glycine has more conformational flexibility that other residues,
so it tends to take up a coiled structure
 The twist of an a helix ensures that critical interactions occur between an
amino acid side chain and the side chain 3-4 resides away
o Positive and negative amino acids form an ion pair 3 resides away
o Two aromatic resides result in stabilization due to hydrophobic effect
 Identity of amino acid residues near the ends play a factor in the stability of
an a helix
o A small electric dipole exists in each peptide bond, aligned through
the hydrogen bonds of the helix
 Results in net dipole along helical axis, increases with length
 o Partial charges of the helix dipole reside on the peptide amino and
carbonyl groups
o Negatively charged amino acids are found near the amino terminus
 Have stabilizing interaction with positive charge of the dipole
 Positively charged amino acid would have destabilizing effect
 Opposite is true at carboxyl-terminal of helical segment
 Five types of constraints affect the stability of an a-helix
o 1. The intrinsic property of an amino acid reside to form an a helix
o 2. The interactions between R groups, particularly those spaces 3-4
residues apart
o 3. The bulkiness of adjacent R groups
o 4. The occurrence of Pro and Gly residues
o 5. The interactions between amino acid residues at the ends of the
helical segment and the electric dipole inherent to the a helix
 The tendency of a segment to form an a helix depends on the identity and
sequence of amino acid resides within the segment

The Beta Conformation Organizes Polypeptide Chains Into Sheets

 The B conformation
o A more extended conformation of polypeptide chains
 Defined by backbone atoms according to dihedral angles
o The backbone is extended into a Zigzag
 Pleated appearance of sheet
o The arrangement of several segments side by side in B conformation
is called a B sheet
 Segments are usually nearby by can also be quite distant or in
different chains
o The R groups of adjacent amino acids protrude from the zigzag in
opposite directions, creating the alternating pattern
o Adjacent polypeptide chains can be parallel or antiparallel
 Having the same or opposite amino-to-carboxyl orientations
 Repeat period is shorter for parallel conformation
 Different hydrogen bonding patterns
 The interstrand hydrogen bonds are in-line in the
antiparallel sheet
 The interstrand hydrogen bonds are distorted/not-in-line
for parallel sheets
B Turns Are Common In Proteins
 Globular proteins, which have a compact folded structure, have some amino
acid residues in turns or loops where the polypeptide chain reverses
directions
o These are the connecting elements that link runs of a helix or b
conformation
o B turns connect the ends of two adjacent segments of an antiparallel b
sheet
 180 degree turn involving four amino acids residues
 Carbonyl oxygen of first residue forming hydrogen bond with
amino-group hydrogen of the fourth
 Peptide groups of the central two residues do not participate
o Several types of B turns have been described, defined by phi and psi
angles of the bonds that link the four amino acids resides that make up
the turn
o Gly and Pro resides often found in b turns
 Gly because is small and flexible
 Pro because peptide bonds involving the imino nitrogen assume
the cis configuration, amenable to a tight turn
o Two types of B turns are the most common
o B turns are often found near the surface of a protein
 The peptide groups of the central two amino acid residues can
hydrogen bond with water
 The gamma turn is less common, a three-residue turn with a hydrogen bond
between the first and third residues

Common Secondary Structures Have Characteristic Dihedral Angles

 The a helix and b conformation are the major repetitive secondary structures
in a wide variety of proteins
 Every secondary structure can be described by the dihedral angles associated
with each residue
o The dihedral angles that define the a helix and the b conformation fall
within a restricted range of sterically allowed structures
o Most values of phi and psi taken from known protein structure fall
into the expected regions, with high concentrations near the a helix
and b conformation values
 Glycine falls outside these regions because it has a small side
chain and can take part in many conformations that are
sterically forbidden for other amino acids
Common Secondary Structures Can Be Assessed by Circular Dichroism
 Structural asymmetry in a molecule gives rise to differences in absorption of
left-handed versus right-handed circularly polarized light
o Measurement of this difference is called circular dichroism (CD)
spectroscopy
 An ordered structure like a protein gives rise to an absorption spectrum that
can have peaks or regions with positive and negative values
o Spectra are obtained in the far UV region
o A signal is obtained when the peptide bond if in a folded environment
o The difference in molar extinction coefficients for left and right
handed circularly polarized light is plotted as a function of wavelength
 The a helix and b conformations have characteristic CD spectra
o Using CD spectra, biochemists can determine whether proteins are
properly folded, estimate the fraction of the protein that is folded in
either of the common secondary structures, and monitor transitions
between the folded and unfolded states

4.3, Protein Tertiary and Quaternary Structures

Tertiary structure: the overall three-dimensional arrangement of all atoms in a

protein

Quaternary structure: arrangement of protein subunits in three-dimensional

complexes

Fibrous proteins: polypeptide chains arranged in long strands or sheets

Globular proteins: polypeptide chains folded into a spherical or globular shaper

Fibrous Proteins Are Adapted for a Structural Function

 Fibrous proteins share properties that give strength and or flexibility to the
structures in which they occur
o The fundamental structural unit is a simple repeating element of
secondary structure
o All fibrous proteins are insoluble in water due to a high concentration
of hydrophobic amino residues in the interior and on the surface
o Hydrophobic surfaces are buried as many polypeptide chains are
packed together to form elaborate complexes
 o Underlying structural simplicity
 A-keratin
o Evolved for strength
o Only in mammals
o Hair, woo, nail claws, quills, horns, hoovers, outer layer of skin
o Part of the family of proteins called intermediate filament (IF)
proteins
 Other IF proteins found in cytoskeletons of animals and share
features exemplified by a-keratins
o Right handed helix
o Crick and Pauling suggested that the a helices were arranged as a
coiled coil
 Two strands of a-keratin, oriented in parallel, are wrapped
about each other to form a super twisted coiled coil
 Super twisting amplifies the strength of the structure
 5.15 to 5.2 A repeating strucutre in hair
 Helical path of supertwists is left handed
 Surfaces where two a helices touch are made up of hydrophobic
amino acid resides
 Their R groups meshed together in an interlocking
pattern
 Permits close packing of the polypeptide chains
o A-Keratin is rich in hydrophobic residues Ala, Val, Leu, Ile, Met, and
Phe
o Individual polypeptide in a-keratin coiled coil had a simple tertiary
structure
 An a-helical secondary structure with its helical axis twisted in
a left-handed superhelix
 The intertwining of the two is the quaternary structure
o Coiled coils of this type are common structural elements in
filamentous proteins and in myosin (muscle protein)
o Many coiled coils can be assembled into large supramolecular
complexes, such as in hair
o The strength of fibrous proteins is enhanced by covalent cross-links
between polypeptide chains in the multi-helical “ropes” and between
adjacent chains in a supramolecular assembly
o In a-keratins, the cross-links stabilizing quaternary structure are
disulfide bonds
 Collagen
o Has also evolved to provide strength
o Found in connective tissue such as tendons, cartilage, the organic
matrix of bone, and the cornea of the eye
o The collagen helix id left handed and has three amino acid residues
per turn
o Collagen is also a coiled coil but with distinct tertiary and quaternary
structures
 Three sperate polypeptides, a-chains, supertwisted about
eachother
 Right handed superhelical twisting
o Many types of vertebrate collagen
 Typically 35% Gly, 11% Ala, 21% Pro and 4-Hyp
o Gelatin is derived from collagen
 Little nutritional value because collagen does not contain many
amino acids essential in the human diet
o Amino acid content is related to the structural constraints uniwue to
the collagen helix
 Generally a repeating tripeptide until, Gly-X-Y
 X is usually Pro
 Y is usually 4-Hyp
 Only Gly residues can be accommodates at very tight junctions
between individual a chains
 Pro and 4-Hyp residues permit the sharp twisting of the helix
o Amino acid sequence and super twisted structure allow very close
packing of its three polypeptides
 4-hydroxyproline has a special role in the structure of collagen
o Tight wrapping of a chains in collagen triple helix provides tensile
strength
 Different associations provide different tensile strengths
o A-chains of collagen molecules and the collagen molecules of fibrils
are cross-linked by unusual types of covalent bonds involving Lys,
HyLys, or His residues present at a few of the X and Y positions
 These links create uncommon amino acid residues
 Increasingly rigid and brittle character of aging connective
tissue results from accumulated covalent cross-links in collagen
fibrils
o Typical mammal has more than 30 variants of collagen
  Genetic defects in collagen structure illustrate the close
relationship between sequence and 3D structure
 Osteogenesis imperfecta
o Abnormal bone formation in babies
o 8 variated with different degrees of severity
 Ehlers-Danlos syndrome
o Loose joints, six variants in humans
 Variates result from substitution of an amino acids
residue with a larger R group (such as Cys or Ser) for a
single Gly reside in an a chain
 Three single-residue substitutions can have a catastrophic
effect on collagen function
o Disrupts the Gly-X-Y repeat that gives collagen its
unique helical structure
o Gly cannot be replaced without substantial
deleterious effects on collagen structure
 Silk Fibroin
o The protein of silk, produced by insects and spiders
o Its polypeptide chains are predominately in the B conformations
o Rich in Ala and Gly residues, permitting close packing of B sheets
and an interlocking arrangement of R groups
o Overall structure is stabilized by extensive hydrogen bonding between
all peptide linkages in the polypeptides of each B sheet and by the
optimization of van der Waals interactions between sheets
o Silk does not stretch because the B conformation is already highly
extended
 The structure is flexible though because the sheets are held
together by numerous weak interactions rather than by covalent
bonds such as the disulfide bonds in a-keratins

Structural Diversity Reflects Functional Diversity in Globular Proteins

 Different segments of the polypeptide chain in globular proteins fold back
on each other
o Generates a more compact shape than in fibrous proteins
o Folding also provides structural diversity needed for proteins to carry
out biological functions
 Enzymes, transport proteins, motor proteins, regulatory proteins,
immunoglobins, and proteins with many other functions
Myoglobin Provided Early Clues About the Complexity of Globular Protein
Structure
 Myosin: a relatively small, oxygen-binding protein of muscle cells
o Stores oxygen and facilitates oxygen diffusion in rapidly contracting
muscle tissues
o Single polypeptide chain of 153 amino acids residues of known
sequence, a single iron protoporphyrin, or heme, group (also found in
hemoglobin, responsible for deep red-brown color)
o Particularly abundant in muscles of diving animals
o Heme is surrounded by protein
o The backbone of myoglobin consists of eight straight segments of a-
helix interrupted by bends, some of which are b turns
o A helix ranges from 7-23 amino acid residues
o All helices are right handed
o More than 70% of amino acid residues are in the a helical regions
o R groups fill up nearly all the space within the folded chain that is not
occupied by backbone atoms
 Many important conclusions were drawn from the structure of myoglobin
o The positioning of amino acid side chains reflects a structure that it
largely stabilized by the hydrophobic effect
 Most hydrophobic R groups are located on the interior
 All but two of the polar R groups are on the outer surface,
hydrated
 So compact, its interior only has room for four molecules of
water
 Dense hydrophobic core is typical of globular proteins
 Weak interactions strengthen and reinforce each other in this
packed environment
 The nonpolar side chains in the core are so close that van
der Waals interactions make a significant contribution to
stabilizing hydrophobic interactions
o All the peptide bonds are in the planar trans configurations
 Three of the four Pro residues are found at bends
 The fourth Pro residue occurs within an a helix, where it creates
a kink necessary for tight helix packing
o The flat heme group rests in a crevice/pocket of the molecule
 The iron atom in the center of the heme group has two bonding
positions perpendicular to the plan of the heme
  One bound to the R group of the His
 The other at the site with O2 binds
 Accessibility of the heme group is highly restricted
 Free heme groups would be rapidly oxidized from the
ferrous form, which is active in the binding of O2, to the
ferric form, which does not bind O2
 As myoglobin structures from many species were resolved, investigators
were able to observe the structural changes associated with the binding of
oxygen or other molecules
o Allowed understanding of correlation between structure and function
o NMR and other techniques supplement x-ray diffraction data
o Sequencing of genomic DNA has identified thousands of genes that
encode proteins of known sequence but unknown function

Globular Proteins Have a Variety of Tertiary Structures

 Each protein has a distinct structure, adapted for its particular biological
function, but they share several important properties with myoglobin
o Each is folded compactly
o Hydrophobic amino acid side chains are oriented toward the interior
o Hydrophilic side chains are on the surface
o Structures are stabilized by a multitude of hydrogen bonds and some
ionic interaction
 Three dimensional structures of globular proteins are considered an
assemblage of polypeptide segments in the a-helix and b conformations,
linked by connecting segments
o Structure can be defined by how the segments stack on one another
and how the segments that connect them are arranged
 To understand a complete three-dimensional structure, we need to analyze
its folding patterns
o Motif: fold (super-secondary structure), a recognizable folding pattern
involving two or more elements of secondary structure and the
connections between them
 Can be very simple, such as two elements of secondary
structure folded against each other
 Ex: b-a-b loop
 Can be very elaborate, involving scores of protein segments
folded together
 Ex: b barrel
  A single large motif may comprise and entire protein
 The segment defined as a motif or fold may or may not be
independently stable
 The coiled coil in a-keratin is a well studied motif
 The arrangement of 8 a helices in myoglobin is replicated in all
globins, called the globin fold
 A motif is not a hierarchical structural element, it is simply a
folding patter
o Domain: a part of a polypeptide that is independently stable or could
undergo movements as a single entity with respect to the entire
protein
 Proteins with more than a few hundred amino acid residues
often fold into two or more domains, sometimes with different
functions
 Domains can often retain their native three-dimensional
structure even when separated from the polypeptide chain
 Each domain may appear as a distinct globular globe
 Excessive contacts between domains make individual
domains hard to discern
 Different domains often have different functions, such as
binding of small molecules or interaction with other proteins
 Small proteins usually have only one domain
 Several rules have emerged from studies of common protein folding patterns
o 1. The hydrophobic effect makes a large contribution to the stability
of protein structures. Burial of hydrophobic amino acid R groups
requires at least two layers of secondary structures
 Simple motifs such as the b-a-b loop create two layers
o 2. Where they occur together in a protein, a helices and b sheets
generally are found in different layers because the backbone of a
segment in the b conformation cannot readily hydrogen bond to an a
helix adjacent to it
o 3. Segments adjacent to each other in the amino acid sequence are
usually stacked adjacent to each other in the folded structure, distant
segments may come together in tertiary structure, but this is not the
norm
o 4. The b conformation is most stable when individual segments are
twisted slightly in a right-handed sends
 Right handed connections tend to be shorter than left handed
ones, making theme easier to form
  Twisting of b sheets also leads to a characteristic twisting of the
structure formed by many such segments together, which forms
the core of many larger structures
 Complex motifs can be built up from simpler ones
o B-a-b loops arranged so that b strands form a barrel creates a
particularly stable and common motif, the a/b barrel
 Each parallel b segment is attached to its neighbored by an a-
helical segment
 All connections are right handed
 The a/b barrel is found in many enzymes, often with a binding
sire in the form of a pocket near one end of the barrel
o Domains with similar folding patterns are said to have the same motif
even though their constituent a helices and b sheets may differ in
length

Some Proteins or Protein Segments Are Intrinsically Disordered

 Some proteins or protein segments lack an ordered structure in solutions
 Some proteins function in the absence of a definable three-dimensional
structure
o Many proteins may be unrestricted or have significant unstructured
segments
 Intrinsically disordered proteins have properties that are distinct from those
of classical, structured proteins
o Lack hydrophobic core and instead are characterized by high densities
of charged amino acids such ad Lys, Arg, and Glu
o Pro residues are also prominent, as they tend to disrupt ordered
structures
 Structural disorder and high charge density can facilitate the function of
some proteins such as spacers, insulators, or linkers in larger structures
o Others are scavengers, binding up ions and small molecules in
solution and serving as reservoirs
 Many intrinsically disordered proteins are at the heart of important protein
interaction networks
o Lack of ordered structure can allow one protein to interact with
multiple partners
o Some wrap around their targets to inhibit action of other proteins
o P27
 Key role in controlling mammalian cell division
 Lacks definable structure in solutions
  Wraps around and inhibits the action of several protein kinases
that facilitate cell division
 Can accommodate to different target proteins
 Human tumor cells have reduced levels of p27
o Some are often present as hubs or scaffolds at the center of protein
networks that constitute signaling pathways
 Interact with different binding partners
 Take on an ordered structure when they interact with other
proteins, but it may vary with different binding partners
 P53
o Critical in the control of cell division
o Contains both structured and unstructured
segments, and the different segments interact with
dozens of other proteins
o An unstructured region at the carboxyl terminus
interacts with four different partners and assumes a
different structure in each of the complexes

Protein Motifs Are the Basis for Protein Structural Classification

 An enormous amount of information about protein structural principles,
function, and evolution is buried in the Protein Data Bank (PDB)
o The Structural Classification of Proteins database (SCOP2) sorts this
information into four categories
 1. Protein relationships
 Features, evolutionary relationships, or other
 2. Structural classes
 All a, all b, a/b, and a + b
 3. Protein types
 Soluble (globular), membrane, fibrous, and intrinsically
unstructured proteins
 4. Evolutionary events
 Traces structural rearrangements and unusual features
o The topology diagram: another way to represent elements of
secondary structure and the relationships among segments of
secondary structure in a protein
 The number of folding patterns is not infinite, about 1,200 different fold or
motifs are represented in the PDB
o Protein tertiary structure is more highly conserved than amino acid
sequence
 o Protein family: proteins with significant similarity in primary structure
or tertiary structure and function
 About 4,000 in the PDB
o Super-families: two or more families that have little similarity in
amino acid sequence by make use of the same major structural motif
and have the same functional similarities
 A protein family may be widespread in all three domains of cellular life
o Many proteins involved in intermediary metabolism and the
metabolism of nucleic acids and proteins
 Other families may be present in only a small group of organisms
o Indicating structure arose more recently

Protein Quaternary Structures Range from Simple Dimers to Large Complexes

 Many multi-subunit proteins have regulatory roles
o The binding of small molecules may affect the interaction between
subunits, causing large changes in protein activity in response to small
changes in concentration of substrate or regulatory molecules
 Separate subunits can take on separate but related functions such as catalysis
and regulation
 Some serve primarily structural roles, such as fibrous proteins
 Some very large protein assemblies are the sites of complex, multistep
reactions
o Ribosomes are the site of protein synthesis, dozens of protein subunits
and RNA molecules are involved
 Multimer: a multi-subunit protein
o Oligomer: only a few subunits
o Asymmetric: nonidentical subunits, quite complicated
o Symmetrical identical subunits or repeating groups of nonidentical
subunits
o Protomer: the repeating structural unit in such a multimeric protein,
whether a single subunit or a group of subunits
 The first oligomeric protein to have its three-dimensional structure
determined was hemoglobin, which contains four polypeptide chains and
four heme prosthetic groups
o Iron atoms are in the ferrous state
o Two a chains
o Two b chains
o Four times as large as myoglobin
 o Subunits are arranged in symmetric pairs, each having on a and one b
subunits
 Can be described as a tetramer or as a dimer of ab protomers

4.4, Protein Denaturation and Folding

Proteostasis: the continual maintenance of the active set of cellular proteins

required under a given set of conditions

Loss of Protein Structure Results in Loss of Function

 Protein structure have evolved to function in particular cellular environments
o Different conditions can result in protein structural changes
o Denaturation: a loss of three-dimensional structure sufficient to cause
loss of function
 Partially folded state
 Most proteins can be denatured by heat
 Complex effects on many weak interactions (primarily
hydrogen bonds)
 If temperature is increased slowly, conformation
generally remains intact until an abrupt loss of structure
occurs over a narrow temperature range
 Loss of function in one part destabilizes other parts
 Very heat-stable proteins of thermophilic bacteria and archaea
have evolved to function at the high temperature of hot springs
 Feature high densities of charged residues on their
surfaces, even tighter hydrophobic packing in their
interiors, and fold rendered less flexible by networks of
ion pairs
 Can also be denatured by pH extremes, certain miscible organic
solvents such as alcohol or acetone, by certain solutes such as
urea and guanidine hydrochloride, or by detergents
 Mild treatment, not covalent bonds are broken
 Urea, organic solvents, and detergents disrupt the
hydrophobic aggregation of nonpolar amino acids
 Urea disrupts hydrogen bonds
 Extremes of pH alter net charge on a protein, causing
electrostatic repulsion and disruption of some hydrogen
bonding
 o Denaturation often leads to protein precipitation, a consequence of
protein aggregate formation as exposed hydrophobic surfaces
associate
 Aggregates are often highly disordered

Amino Acid Sequence Determines Tertiary Structure

 Proof came from experiments showing that denaturation of some proteins is
reversible if returned to conditions in which the native conformation is stable
o Renaturation
 Denaturation and renaturation of ribonuclease
o Denatures completely in concentrated urea solution in the presence of
a reducing agent
 Reducing agent cleaved the four disulfide bonds to yield eight
cys residues
 Urea disrupts the stabilizing hydrophobic effect
 Entire peptide is freed form its folded conformation
o Complete loss of catalytic activity
o When urea and the reducing agent are removed, the protein
spontaneously refolds into its current tertiary structure
 Full restoration of its catalytic activity
 The Anfinsen experiment proved that the amino acid sequence contains all
the information required to fold the chain into tis native, three-dimensional
structure
o Many require some assistance

Polypeptides Fold Rapidly by a Stepwise Process

 Proteins are assembled from amino acids at a very high rate
o Major folding pathways are hierarchical
 Local secondary structures form first
 Certain amino acid sequences fold readily into a helices
of b sheets
 Ionic interactions can play important roles in these early
steps
 Longer-range interactions between two elements of secondary
structure that come together to form stable folded structures
 Hydrophobic effect plays a significant role
 Process continues until complete domains form and the entire
polypeptide is folded
 o Proteins dominated by close-range interactions tend to fold faster than
proteins with more complex folding patterns and with many long-
range interactions between different segments
 Domains near the amino terminus may fold before the entire
polypeptide has been assembled in larger proteins with multiple
domains
 The folding process can be viewed as a kind of free-energy funnel
o Unfolded states are characterized by a high degree of conformational
entropy and relatively high free energy
o As folding proceeds, the narrowing of the funnel reflects the decrease
in the conformational space that must be searched as the protein
approaches its native state
o At the bottom of the funnel, an ensemble of folding intermediates has
been reduces to a single native conformation
o The funnels can have a variety of shapes, depending on the
complexity of the folding pathway, the existence of semi-stable
intermediates, and the potential for particular intermediates to
assemble into aggregates of misfolded proteins
 Thermodynamic stability is not evenly distributed over the structure of a
protein
o Regions of high and low stability
o Regions of low stability may allow a protein to alter its conformation
between two or more states
o Variations in the stability of regions within a protein are often
essential to protein function
o Intrinsically disordered proteins or protein segments do not fold at all

Some Proteins Undergo Assisted Folding

 Chaperons: proteins that interact with partially folded or improperly folded
polypeptides, facilitating correct folding pathways or providing
microenvironments in which folding can occur
o Two major families
 Hsp70
 Family of proteins with molecular weight near 70,000
 More abundant in cells stressed by elevated temperatures
 Bind to regions of unfolded polypeptides that are rich in
hydrophobic residues
 “protect” proteins subject to denaturation by heat & new
molecules being synthesized
  Block the folding of certain proteins that must remain
unfolded until they are across the membrane
 Some facilitate the quaternary assembly of oligomeric
proteins
 Binds to and releases polypeptides in a cycle that uses
energy from ATP hydrolysis and involves several other
proteins
 The binding of an unfolded polypeptide by an Hsp70
chaperone may break up a protein aggregate or prevent
the formation of a new one
 When the bond polypeptide is released, it has a chance to
resume folding to its native structure
 If folding does not occur rapidly enough, the polypeptide
may be bound again and the process repeated
o Alternatively, the Hsp70-bound polypeptide may
be delivered to a chaperonin
 Chaperonins
 Elaborate protein complexes required for the folding of
some cellular proteins that do not fold spontaneously
 Many E Coli proteins require the resident chaperonin
system, GroEL/GroES
o Eukaryotic system is called Hsp60
 An unfolded protein is bound to an exposed hydrophobic
surface near the apical end of on GroEL chamber
 The protein is them trapped within the chamber when is
is transiently capped by the “lid”
 GroEL undergoes conformational changes, coupled to
slow ATP hydrolysis, which regulates the biding and
release of GroES
 Inside the chamber, a protein has about 10 seconds to
fold, the time required for the bond ATP to hydrolyze
 The protein is released when the GroES cap dissociates,
but can rebind rapidly for another round if folding has
not been completed
 The two chambers in the GroEL complex alternate in
binding and releasing unfolded polypeptide substrates
 The Hsp60 system utilizes a similar process to fold
proteins
 o In the place of the GroES lid, protrusions from the
apical domains of the subunits flex and close over
the chamber
o The ATP hydrolytic cycle is also slower in the
Hsp60 complexes, giving the constrained proteins
more time to fold
o The folding pathways of some proteins require two enzymes that
catalyze isomerization reactions
 Protein disulfide isomerase (PDI) is a widely distributed
enzyme that catalyzes the interchange, or shuffling, of disulfide
bonds until the bonds of the native conformation are formed
 PDI catalyzes the elimination of folding intermediates
with inappropriate disulfide cross-links
 Peptide prolyl cis-trans isomerase (PPI) catalyzes the
interconversion of the cis and trans isomers of peptide bonds
formed by Pro residues, which can be a slow step in the folding
of proteins that contain some Pro peptide bonds in the cis
configuration

Defects in Protein Folding Provide the Molecular Basis for a Wide Range of
Human Genetic Disorders
 Protein misfolding is a substantial problem in all cells and can contribute to
the development of serious disease
o Type 2 diabetes, Alzheimer disease, Huntington disease, and
Parkinson disease are all associated with a misfolding mechanism
 A soluble protein that is normally secreted from the cell is
secreted in a misfolded state and converted into an insoluble
extracellular amyloid fiber
 The diseases are collectively referred to as amyloidosis
 Highly ordered and unbranched fibers
 High degree of b-sheet structure
 B segments are oriented perpendicular to the axis of the
fiber
 In some amyloid fibers, the overall structure includes two
layers of b sheet, such as that shown for amyloid-b peptide
o Many proteins can take on the amyloid fibril structure as an
alternative to their normal folded conformations
 Most of these proteins have a concentration of aromatic amino
acid residues in a core region of b sheet or a helix
  The proteins are secreted in an incompletely folded formation
 The core folds into a b sheet before the rest of the protein folds
correctly, and the b sheets from two or more associate to begin
forming an amyloid fibril
 The fibril grows in the extracellular space
 Other parts of the protein then fold differently, remaining on the
outside of the b-sheet core in the growing fibril
o Because most of the protein molecules fold normally, the onset of
symptoms in the amyloidosis is often very slow
 If a person inherits a mutation such as substitution with an aromatic residue
at a position that favors formation of amyloid fibrils, disease symptoms may
being at an earlier age
 In eukaryotes, proteins destined for secretion undergo their initial folding in
the ER
o When stress conditions arise, unfolded proteins can accumulate
o Triggers the unfolded protein response (UPR)
 The transcriptional regulators that constitute the UPR bring the
various systems into alignment by increasing chaperonin
concentration or decreasing the rate of protein synthesis
o Amyloid aggregates formed before the UPR can come into play, may
be removed
 Some are degraded by autophagy
 The aggregates are encapsulated in the membrane, and
then degraded with a cytosolic lysosome
o Misfolded proteins can be degraded by a system of proteases called
the ubiquitin-proteasome system
 Some amyloidosis are systemic, involving many tissues
o Primary systemic amyloidosis is caused by deposition of fibrils
consisting of misfolded immunoglobin light chains, or fragments of
light chains derived from proteolytic degradation
 Mean onset age of 65
 Fatigue, hoarseness, swelling, weight loss, death within a year
of diagnosis
 Kidney and heart are often most affected
 Can also be associated with other types of disease
o Secondary systemic amyloidosis
 Wide range of symptoms depending on the organs initially
affected
 Fatal within a few years
 o More than 80 amyloidosis are associated with mutations in
transthyretin
 A variety of mutations in this protein lead to amyloid
deposition concentrated around different tissues, thus producing
different symptoms
 Also associated with inherited mutations in the lysosome,
fibrinogen A a chain, and apolipoproteins
 Some amyloid diseases are associated with particular organs
o The amyloid-prone protein is generally secreted only be the affected
tissue, and its locally high concentration leads to amyloid deposition
around that tissue
o One common site of deposition is near the pancreatic islet b cells,
responsible for insulin secretion and regulation of glucose metabolism
o With progressive loss of these cells, glucose homeostasis is affected
and eventually the condition matures into type 2 diabetes mellitus
 The amyloid deposition diseases that trigger neurodegeneration are a special
class of localized amyloidoses
o Alzheimer disease is associated with extracellular amyloid deposition
by neurons, involving the amyloid-b peptide
o When it is part of the larger protein, the peptide is composed of two a-
helical segments spanning the membrane
o When the external and internal domains are cleaved off by proteases,
the relatively unstable amyloid-b peptide leaves the membrane and
loses it’s a-helical structure
o It can then take the form of two layers of extended parallel b sheets
which can slowly assemble into amyloid fibrils
o Deposits of these amyloid fibers seems to be the primary cause of
Alzheimer’s disease
o A second type of amyloidlike aggregation, involving the protein tau,
also occurs intracellularly in neurons of people with Alzheimer
disease
 Several other neurodegenerative conditions involve intracellular aggregation
of misfolded proteins
o Parkinson’s Disease
 The misfolded form of the protein a-synuclein aggregates into
spherical filamentous masses called Lewy bodies
o Huntington disease
 Involves the proteins huntingtin, which has a long
polyglutamine repeats
  In some, this repeat is longer than normal and a more subtle
type of intracellular aggregation occurs
o Symptoms are highly suppressed if expression of the Hsp70 chaperon
is increased
 Protein misfolding doesn’t need to lead to amyloid formation to cause
serious disease
o Cystic fibrosis is caused by defects in a membrane-bound protein
called CFTR which acts as a channel for chloride ions
 The most common CF causing mutation is the deletion of a Phe
residue at position 508 in CFTR, which causes improper protein
folding
 Most of the protein is then degraded and its normal function is
lost
 Many of the disease-related mutations in collagen also cause
defective folding
 A particularly remarkable kind of protein misfolding is seen in
the prion diseases

Lecture: Working With Proteins

 Review techniques for working with protein separation
 Homogenization if working with liquids
 Purification methods of proteins
 Size chromatography -
 Ion exchange chromatography -
o Cation -
o Anion –
 Role of SDS gel –
 Mass spectrometry
o When would you use it?
o what type of job can it do?
o Get precise MW, do sequencing with tandem MS, study modifications
such as phosphorylation or prenylation
o Problem - working with big molecules
 Need to be in gas phase – hard with big molecules
 Need to find way to evaporate and ionize molecules
 MALDI – matric assisted laser desorption ionization
o Sample of plate, short laser evaporates and ionizes
sample
  ESI – electrospray ionization
o Solution is nebulized from thin capillary connected
to high voltage
